Vaccine therapy for treatment of endometrial and ovarian cancer

ABSTRACT

The present invention provides methods of preventing or reducing the risk of recurrence of endometrial and ovarian cancers which express low levels of folate binding protein (FBP) by administration of a vaccine containing an E39 peptide.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/609,709, which was filed May 31, 2017, which claims the benefit ofthe priority date of U.S. Provisional Application No. 62/343,675 whichwas filed May 31, 2016. The contents of which are hereby incorporated byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 31, 2018, isnamed RXJ-024CN_Sequence_Listing.txt and is 4,488 bytes in size.

FIELD OF THE INVENTION

The present invention is directed to the use of peptide based cancervaccines.

BACKGROUND OF THE INVENTION

Endometrial cancer is the most common gynecologic cancer in the UnitedStates (Senol, et al., Int. J. Clin. Exp. Pathol. 2015; 8(5):5633-5641).Ovarian cancer, though less common, is the most common cause of deathamongst gynecologic cancers. Due to the nonspecific symptoms of ovariancancer, >70% of patients will present with late stage disease and, evenwith aggressive regimens, fewer than 40% of women with ovarian cancerare cured after standard of care treatment. Endometrial cancer has amuch better prognosis in general because of earlier diagnosis, but evenin early stage disease, 10-15% of patients will experience a recurrence.The serous variant of endometrial cancer is particularly aggressive,often presenting with evidence of metastasis (Black et al., Int J of GynCancer. 2016; 26(1):133-140), and having a clinical course more similarto that of ovarian carcinoma than typical endometrial cancer. Althoughonly responsible for 3-10% of all endometrial cancers, this subtypeleads to a high risk of recurrence, and up to 40% of endometrial cancerdeaths.

Current treatments for endometrial and ovarian cancer include surgery,chemotherapy, and radiation, but these are insufficient in somepatients, particularly those with unfavorable histology and advancedtumor stages. In addition, several novel medications have been tested inpatients with Stage II or higher endometrial and ovarian cancers,including those that inhibit biochemical processes (anti-angiogenesis,DNA repair mechanisms, mTOR pathway, etc.) and monoclonal antibodies(anti-CA125 and anti-human epithelial cell adhesion molecule—EpCAM)(Ziebarth et al., Molecular/Genetic Therapies in Ovarian Cancer: FutureOpportunities and Challenges. In: Clinical Obstetrics and Gynecology.2012. Baltimore, Md.; Tse et al. Ann Oncol 2014; 25(2):322-331).

The folate binding protein (FBP), also known as folate receptor-alpha(FRa), is a tumor associated antigen (TAA) common to both endometrialand ovarian cancer with up to 80-90 fold higher expression in malignantcells compared to normal cells (Li et al., J Nucl Med. April 1996;37(4):665-672; Weitman et al., Cancer Res. Jun. 15, 1992;52(12):3396-3401). FBP levels coincide with increased folate uptake,which is necessary for DNA production in rapidly replicating cells. As aresult, its over-expression has been associated with higher grade andstage of endometrial and ovarian cancers (Toffoli et al., Int J Cancer.Apr. 22, 1997; 74(2):193-198). High levels of FBP have also beenassociated with the more aggressive serous variant of endometrial cancer(Dainty et al., Gyn Onc. 2007; 105(3):563-570). Furthermore, FBP mayalso be associated with failure to respond to platinum-basedchemotherapy and reduced survival in ovarian cancer patients withresidual disease after surgical treatment (Toffoli et al., Int J Cancer.Apr. 17, 1998; 79(2):121-126).

Highlighting the importance of this TAA, there have already beensignificant efforts toward developing novel FBP-targeted therapies,including a monoclonal antibody, farletuzumab, alone or in conjugates todeliver radionuclides, FRa-targeted T cells and stimulatory cytokines tomalignant tissues. Additionally, immunotherapy methods forFRα-expressing cancers currently being investigated include the use offolate-localized molecules to enhance cancer immunogenicity, techniquesto raise FRa-specific immunity via viral vector, as well as multiplevaccine strategies including modified whole tumor cells, as well as DNA,dendritic cell and multi-peptide peptide vaccines (reviewed for exampleby Clifton et al., Human Vaccines 7:183-190, 2011).

Previous studies have shown that FRa contains naturally processedimmunogenic peptides that are recognized by tumor-associatedlymphocytes, and that two specific FRa-derived immunogenic peptides, E39(FRα 191-199) and E41 (FRα 245-253), are capable of enhancingtumor-associated lymphocyte (TAL) proliferation and anti-tumor function(Peoples et al., Ann Surg Oncol 1998; 5:743-50). The HLA-A2 restrictedE39 peptide has also been shown to enhance tumor-associated lymphocyteproliferation and anti-tumor function in pre-clinical trials (Kim etal., Anticancer Res. July-August 1999; 19(4B):2907-2916; Peoples et al.,Ann Surg Oncol 1998; 5:743-50; Peoples et al., Clin Cancer Res 1999;5:4214-23).

However, despite some promising results with various regimens,durability of response remains limited. Given the propensity forrecurrence in patients with aggressive forms of endometrial cancer, atreatment with better long-term efficacy is needed.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, that a vaccinecontaining the E39 peptide is effective in preventing recurrence ofaggressive forms of endometrial and ovarian cancers which express lowlevels of FRa.

Accordingly, in one aspect, a method of preventing or reducing the riskof recurrence of an aggressive endometrial or ovarian cancer in a humansubject is provided. The method comprises administering to the subject avaccine comprising at least about 1.0 mg of a peptide comprising theamino acid sequence of SEQ ID NO: 1), and human granulocyte macrophagestimulating factor (GM-CSF) as an adjuvant every three to four weeksuntil protective immunity is established.

In some embodiments, the cancer is a serous variant of endometrialcancer. In one embodiment, the cancer is ovarian carcinoma. In oneembodiment, the endometrial cancer is serous uterine cancer. Inparticular embodiments, the endometrial or ovarian cancer ischaracterized as a Stage II or higher cancer. In related embodiments,the endometrial or ovarian cancer is characterized as having a lowexpression level of FRa, for example, an immunohistochemistry (IHC)rating of 0 or 1+.

In some embodiments, the human subject has been previously treated(e.g., received a primary treatment) with one or more cancer therapies.In some embodiments, the primary treatment includes surgery,chemotherapy or a combination thereof. In one embodiment, the subjecthas no evidence of disease (NED) after completion of the primarytreatment. In one embodiment, the peptide vaccine is administered withinabout three months after the primary treatment is completed.

In some embodiments, the vaccine is administered by inoculation orinjection. In one embodiment, the vaccine is administered by intradermalinjection in one dose or in one or more split dosages. In someembodiments, the vaccine is administered as split dosages that areadministered substantially concurrently. In related embodiments, thesplit dosages are administered at one site or at different sites. Inother related embodiments, the split dosages are administered at least 5cm apart.

In some embodiments, the vaccine is administered every three to fourweeks for a period of six months. In some embodiments, eachadministration of the vaccine occurs at the same site or at differentsites. In one embodiment, each administration of the vaccine occurs inthe same lymph node draining area.

In some embodiments, the GM-CSF is present in the vaccine in an amountbetween about 0.01 to about 0.5 mg GM-CSF. In one embodiment, thevaccine contains about 0.250 mg of GM-CSF. In one embodiment, thepeptide consists of the amino acid sequence of SEQ ID NO: 1. In anotherembodiment, vaccine comprises a 1.0 of a peptide consisting of the aminoacid sequence of SEQ ID NO: 1, and about 0.250 GM-CSF.

In a related aspect, a method for inducing and maintaining an immuneresponse to an aggressive endometrial cancer in a human subject isprovided. The method comprises (a) administering to the subject avaccine comprising at least about 1.0 mg of a peptide comprising theamino acid sequence of SEQ ID NO: 1), and human granulocyte macrophagestimulating factor (GM-CSF) as an adjuvant every three to four weeksuntil protective immunity is established, and (b) administering abooster composition comprising a peptide comprising an amino acidselected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, andan adjuvant about six months, about twelve months or about one yearafter completion of the primary immunization schedule.

In one embodiment, the booster composition contains a peptide consistingof the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In oneembodiment, the booster composition comprises a peptide consisting ofthe amino acid sequence of SEQ ID NO: 1. In one embodiment, the boostercomposition comprises an peptide consisting of the amino acid sequenceof SEQ ID NO: 2.

In some embodiments, the booster composition comprises about 0.5 toabout 1.0 mg of the peptide. In some embodiments, the adjuvant in thebooster composition is GM-CSF in an amount between about 0.01 mg toabout 0.5 mg GM-CSF. In one embodiment, the booster compositioncomprises about 0.250 GM-CSF.

Also provided are kits that include the peptide vaccines used in themethods of the invention. In one embodiment, the kit contains the E39peptide vaccine in unit dosage form of about 1.0 mg. Other features andadvantages of the instant disclosure will be apparent from the followingdetailed description and examples, which should not be construed aslimiting.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow diagram depicting the treatment protocol for patientsenrolled in phaseI/IIa clinical trial.

FIG. 2 is a bar graph depicting the maximum local systemic toxicityexperienced by patients vaccinated with E39. There was a statisticallysignificant different between the local toxicity experienced by the OPTvs nOPT group, p=0.04. No significant difference was noted withinsystemic toxicity.

FIG. 3 is a bar graph depicting the local reactions to vaccination andDTH response before and after the primary vaccination series accordingto dosing cohorts expressed as the orthogonal mean of the reaction sizeand expressed as means. The average size of induration to E39 prior tovaccination was 5.7±1.5 mm compared to 10.3±2.8 mm post-vaccination(p=0.06). OPT patients had a statistically significant increase inpre-vaccination versus post-vaccination DTH (3.8±2.0 mm vs 9.5±3.5 mm,p=0.03), while nOPT patients experienced a smaller increase in pre- vspost-vaccination, which was not statistically significant (7.8±2.1 mm vs11.3±4.8 mm, p=0.28).

FIG. 4 depicts the 2-year estimated Kaplan Meier disease-free survivalcurves for vaccinated patients was 43% (95% confidence interval (CI):18-66%) versus 33.6% (95% CI: 13-56%) in control patients (p=0.36). Thevaccinated patients experienced a 31% reduction in relative recurrencerisk regardless of dose.

FIG. 5 depicts the 2-year estimated Kaplan Meier disease-free survivalcurves for the subgroup analysis performed based on E39 peptide dosing.The 2-year estimated DFS indicated a significant survival advantage forthe OPT group at 85.7% (95% CI: 54-96%) compared to controls at 33.6%(95% CI: 13-56%) and the nOPT group at 20.8% (95% CI: 4-47%). Comparingthe OPT and control groups, there was an 83% reduction in relative riskof recurrence.

FIG. 6 depicts the overall disease-free survival curves for control andE39 treated patients.

FIG. 7 (A) depicts the overall survival curves for control and E39treated patients with IHC for FBP protein expression of 0 to 1+; (B)depicts the overall survival curves for control and E39 patients withIHC for FBP protein expression of 2-4+.

FIG. 8 (A) depicts the overall curves for control patients with IHC 0,1+ vs. 2-4+; (B) depicts the overall survival curves for E39 treatedpatients with IHC 0, 1+ vs. 2-4+

FIG. 9 depicts the overall survival curves for untreated controlpatients, patients treated with less than 1000 mcg E39, and patientstreated with 1000 mcg E39.

FIG. 10 depicts the overall survival curves for patients with FBP IHC 0or 1+ treated with less than less than 1000 mcg E39, or patients treatedwith 1000 mcg E39.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In order that the present description may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art, andconventional methods of immunology, protein chemistry, biochemistry,recombinant DNA techniques and pharmacology are employed.

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. The use of “or”or “and” means “and/or” unless stated otherwise. Furthermore, use of theterm “including” as well as other forms, such as “include”, “includes”,and “included”, is not limiting.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration and the like, is encompassesvariations of up to ±10% from the specified value. Unless otherwiseindicated, all numbers expressing quantities of ingredients, propertiessuch as molecular weight, reaction conditions, etc., used herein are tobe understood as being modified by the term “about”.

The term “antigen” as used herein is defined as an entity which elicitsan immune system response. The term herein may be abbreviated to “Ag.”

“Booster” refers to a dose of an immunogen administered to a patient toenhance, prolong, or maintain protective immunity and to overcome thedown-regulation of T-cell responses mediated by regulatory T-cells.

The term “cancer” as used herein is defined as a tissue of uncontrolledgrowth or proliferation of cells, such as a tumor. As used herein, theterm includes pre-malignant as well as malignant cancers. “Ovariancancer” includes primary peritoneal or fallopian tube malignancies.

The term “endometrial cancer” as used herein refers generally to cancerarising from the lining (endometrium) of the uterus. An “aggressiveendometrial cancer” can be a serous variant of endometrial cancer, orcharacterized based on histology as intermediate or high grade (grade 2or 3), or stage II or higher.

The term “deplete” in the context the therapeutic methods disclosedherein refers to a reduction in the number of, or elimination of cancercells expressing folate receptor alpha.

The terms “effective treatment” or “positive therapeutic response” areused interchangeably to refer to a course of treatment producing abeneficial effect, e.g., amelioration of at least one symptom of adisease or disorder. A beneficial effect can take the form of animprovement over baseline, i.e., an improvement over a measurement orobservation made prior to initiation of therapy according to the methodsprovided herein, including slowing, stopping or reversing theprogression of an endometrial cancer in a subject at any clinical stage,as evidenced by a decrease or elimination of a clinical or diagnosticsymptom of the disease after administration of the FRα peptide vaccinein accordance with the methods described herein. Effective treatmentmay, for example, decrease in tumor size, decrease the presence ofcirculating tumor cells, reduce or prevent metastases of a tumor, slowor arrest tumor growth and/or prevent or delay tumor recurrence orrelapse.

“Effective amount,” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound orcomposition, used according to the methods provided herein that iseffective to inhibit the occurrence or ameliorate one or more clinicalor diagnostic symptoms of a FRα expressing cancer, as determined by anymeans suitable in the art. Such results can include, but are not limitedto, an increase in FRa-specific cytotoxic T-lymphocytes (CTLs), adecrease in circulating tumor cells, a decrease in tumor size, thecessation of tumor growth and/or the prevention or delay of tumorrecurrence or relapse in a subject.

The term “epitope” as used herein is defined as a short peptide derivedfrom a protein antigen which binds to an MHC molecule and is recognizedby a particular T cell. An epitopic core sequence, as used herein, is arelatively short stretch of amino acids that is “complementary” to, andtherefore will bind, receptors on CTLs. It will be understood that inthe context of the present disclosure, the term “complementary” refersto amino acids or peptides that exhibit an attractive force towards eachother.

As used herein, the term “an epitope(s) that is immunologicallyrecognized by a CTL” is intended to refer to a peptide or proteinantigen which includes a primary, secondary or tertiary structuresimilar to an epitope located within a FRα polypeptide. The level ofsimilarity will generally be to such a degree that the same populationof CTLs will also bind to, react with, or otherwise recognize, thecross-reactive peptide or protein antigen.

The term “folate receptor α” (also referred to as FRα, FRA, FR-alpha,FOLR-1, folate binding protein, FBP, FOLR1, LK26 trophoblastic antigenand GP38) refers to the alpha isoform of the high affinity receptor forfolate. Membrane bound FRα is attached to the cell surface by a glycosylphosphtidylinositol (GPI) anchor, recycles between extracellular andendocytic compartments and is capable of transporting folate into thecell. FRα is expressed in a variety of epithelial tissues includingthose of the female reproductive tract, placenta, breast, kidney,proximal tubules, choroid plexus, lung and salivary glands. Solubleforms of FRα may be produced by the action of proteases orphospholipase. As used herein “soluble FRα” refers to FRα that is notmembrane bound and is present in biological fluids, e.g., serum orurine. For example, soluble FRα may be shed, secreted or exported fromnormal or cancerous cells into biological fluids.

(SEQ ID NO: 3)tcaaggttaa acgacaagga cagacatggc tcagcggatg acaacacagc tgctgctcct  60tctagtgtgg gtggctgtag taggggaggc tcagacaagg attgcatggg ccaggactga 120acttctcaat gtctgcatga acgccaagca ccacaaggaa aagccaggcc ccgaggacaa 180gttgcatgag cagtgtcgac cctggaggaa gaatgcctgc tgttctacca acaccagcca 240ggaagcccat aaggatgttt cctacctata tagattcaac tggaaccast gtggagagat 300ggcacctgcc tgcaaacggc atttcatcca ggacacctgc ctctacgagt gctcccccaa 360cttggggccc tggatccagc aggtggatca gagctggcgc aaagagcggg tactaaacgt 420gcccctgtgc aaagaggact gtgagcaatg gtgggaagat tgtcgcacct cctacacctg 480caagagcaac tggcacaagg gctggaactg gacttcaggg tttaacaagt gcgcagtggg 540agctgcctgc caacctttcc atttctactt ccccacaccc actgttctat acaatgaaat 600ctggactcac tcctacaagg tcagcaacta cagccgaggg agtggccgct gcatccagat 660gtggttcgac ccagcccagg gcaacccsaa tgagaagatg gegaggttct atgctgcagc 720catgagtggg gctgggccct gggcagcctg gcctttcctg cttagcctgg ccctaatgct 780gctgtggctg ctcagctgac ctccttttac cttctgatac ctggaaatcc ctgccctgtt 840cagccccaca gctcccaact atttggttcc tgctccatgg tcgggcctct gacagccact 900ttgaataaac cagacaccgc acatgtgtct tgagaattat ttggaaaaaa aaaaaaaaaa 960 aa962 (SEQ ID NO: 4)Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp ValAla Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr GluLeu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro GlyPro Glu Asp Lys Leu His His Gln Cys Arg Pro Trp Arg Lys Asn AlaCys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser TyrLeu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala CysLys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro AsnLeu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu ArgVal Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp GluAsp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly TrpAsn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys GlnPro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu IleTrp Thr His Ser Tyr Lys Val Her Asn Tyr Ser Arg Gly Ser Gly ArgCys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu GluVal Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp AlaAla Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu Ser

“Free of cancer” or “disease free” or NED (No Evidence of Disease) meansthat the patient is in clinical remission induced by treatment with thecurrent standard of care therapies. By “remission” or “clinicalremission,” which are used synonymously, it is meant that the clinicalsigns, radiological signs, and symptoms of cancer have beensignificantly diminished or have disappeared entirely based on clinicaldiagnostics, although cancerous cells can still exist in the body. Thus,it is contemplated that remission encompasses partial and completeremission. The presence of residual cancer cells can be enumerated byassays such as CTC (Circulating Tumor Cells) and can be predictive ofrecurrence.

The term “immune response” as used herein refers to a cellular immuneresponse, including eliciting stimulation of T lymphocytes, macrophages,and/or natural killer cells.

The term “immunity” as used herein is defined as the ability to provideresistance to a tumor resulting from exposure to an antigen that is afolate binding protein epitope.

The term “inhibit” or “inhibition” means to reduce by a measurableamount.

The “level” of a specified protein refers to the amount of protein in asample as determined using any method known in the art for measuringprotein levels, including electrophoresis, capillary electrophoresis,high performance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, fluid or gel precipitationreactions, absorption spectroscopy, colorimetric assays,spectrophotmetric assays, flow cytometry, immmunodiffusion, solutionphase assay, immunoelectrophoresis, Western blotting, radioimmunoassay(MA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescentassays and electrochemiliminescence immunoassays.

“Peptide” refers to any peptide comprising two or more amino acidsjoined by peptide bonds or modified peptide bonds (e.g., peptideisosteres). Peptides can contain amino acids other than the 20 naturallyoccurring nucleic acid encoded amino acids, and include amino acidsequences modified either by natural processes, such aspost-translational processing, or by chemical modification techniqueswhich are well known in the art. Modifications can occur anywhere in apeptide, including the peptide backbone, the amino acid side-chains andthe amino or carboxyl termini. It will be appreciated that the same typeof modification can be present in the same or varying degrees at severalsites in a given peptide. Also, a given polypeptide can contain manytypes of modifications. Polypeptides can be branched as a result ofubiquitination, and they can be cyclic, with or without branching.Cyclic, branched and branched cyclic polypeptides can result fromnatural posttranslational processes or can be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination.

The term “prevent” refers to any success or indicia of success in theforestalling of cancer recurrence or relapse in a subject in clinicalremission, as measured by any objective or subjective parameter,including the results of a radiological or physical examination, or inthe level of circulating tumor cells.

“Protective immunity” or “protective immune response” means that asubject mounts an active immune response to an immunogenic component ofan antigen such as the FRα antigens described and exemplified herein,such that upon subsequent exposure to the antigen, the subject's immunesystem is able to target and destroy cells expressing the antigen,thereby decreasing the incidence of morbidity and mortality associatedwith cancer in the subject. For example, protective immunity in thecontext of the present methods is conferred at least partially by Tlymphocytes.

“Optimal biologic dose (OBD)” is defined as the minimum dose of a drugthat gives the most optimal and lasting in vivo response withoutclinically unacceptable toxicity. For example, the OBD of a peptidevaccine is the amount that gives the most optimal and lasting in vivoimmunologic response to the peptide.

“Relapse” or “recurrence” or “resurgence” are used interchangeablyherein, and refer to the radiographic diagnosis of return, or signs andsymptoms of return of cancer after a period of improvement or remission.

The term “sample” refers to a collection of fluids, cells or tissuesisolated from a subject. Biological fluids are typically liquids atphysiological temperatures and may include naturally occurring fluidspresent in, withdrawn from, expressed or otherwise extracted from asubject or biological source. Examples of biological fluids includeblood, serum, serosal fluids, plasma, lymph, urine, cerebrospinal fluid,saliva, ocular fluids, cystic fluid, tear drops, feces, sputum, mucosalsecretions, vaginal secretions, gynecological fluids, ascites fluidssuch as those associated with non-solid tumors, fluids of the pleural,pericardial, peritoneal, abdominal and other body cavities, fluidscollected by bronchial lavage and the like.

The term “control sample”, as used herein, refers to any clinicallyrelevant control sample, including, for example, a sample from a healthysubject or a sample made at an earlier timepoint from the subject to beassessed. For example, the control sample can be a sample taken from thesubject prior to onset of cancer, at an earlier stage of disease, orbefore the administration of treatment or of a portion of treatment.

The term “subject” or “patient” are used interchangeably herein andrefer to a mammal such as a human, mouse, rat, hamster, guinea pig,rabbit, cat, dog, monkey, cow, horse, pig and the like.

As used herein, “substantially purified” refers to a vaccine component(e.g, a protein, polypeptide, or peptide) that has a level of puritywhere the compound is substantially free from other chemicals,biomolecules or cells. For example, a substantially purified vaccinecomponent is about 60%, about 70%, about 80%, about 90%, about 95%,about 99% or more a free of the other components of the manufacturingprocess (e.g., cellular extract or reagents of chemical synthesis).Various techniques suitable for use in chemical, biomolecule orbiological purification, well known to those of skill in the art, may beapplicable to preparation of a vaccine component of the presentinvention. These include, for example, precipitation with ammoniumsulfate, polyethylene glycol (PEG), antibodies and the like or by heatdenaturation, followed by centrifugation; fractionation, chromatographicprocedures, including but not limited to, partition chromatograph (e.g.,paper chromatograph, thin-layer chromatograph (TLC), gas-liquidchromatography and gel chromatography) gas chromatography, highperformance liquid chromatography, affinity chromatography,supercritical flow chromatography ion exchange, gel filtration, reversephase, hydroxylapatite, lectin affinity; isoelectric focusing and gelelectrophoresis (see for example, Sambrook et al. 1989; and Freifelder,Physical Biochemistry, Second Edition, pages 238-246, incorporatedherein by reference).

The term “variant” as used herein is defined as a modified or alteredform of a wildtype sequence. The term “folate binding protein variant”as used herein is defined as a folate binding protein and peptidesthereof which are preferably recognized by helper T cells or cytotoxic Tcells and may be naturally derived, synthetically produced, geneticallyengineered, or a functional equivalent thereof, e.g. where one or moreamino acids may be replaced by other amino acid(s), (e.g., or non-aminoacid(s) which do not substantially affect function. In some embodiments,the variant may contain an altered side chain for at least one aminoacid residue.

Various aspects described herein are described in further detail in thefollowing subsections.

Peptides

The peptides used in the vaccine composition according to the methods ofthe invention comprise the FRα epitope E39 having the amino acidsequence EIWTHSYKV (SEQ ID NO: 1).

Peptides for use in the vaccine composition in accordance with themethods provided herein will generally be on the order of 9 to 20 aminoacids in length, and more preferably about 9 to about 15 amino acids inlength, and comprise the epitope core sequence of E39 (SEQ ID NO: 1). Incertain embodiments, the peptides are at least about 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 amino acids in length. In one particularembodiment, the peptide in the vaccine composition consists of the aminoacid sequence of E39.

The peptides used in the booster composition according to the methods ofthe invention comprise the E39 peptide amino acid sequence (SEQ ID NO:1), or the amino acid sequence of a modified FRα eptitope such as E39′having the amino acid sequence EIWTFSTKV (SEQ ID NO: 2). In oneembodiment, the peptide in the booster compositions consists of theamino acid sequence of E39 (SEQ ID NO: 1), and E39′ (SEQ ID NO: 2).

In general, due to the relative stability of peptides, they may bereadily stored in aqueous solutions for fairly long periods of time ifdesired, e.g., up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of antigenic activity.However, where extended aqueous storage is contemplated it willgenerally be desirable to include agents including buffers such as Trisor phosphate buffers to maintain a pH of about 7.0 to about 7.5.Moreover, it may be desirable to include agents which will inhibitmicrobial growth, such as sodium azide or Merthiolate. For extendedstorage in an aqueous state it will be desirable to store the solutionsat 4° C., or frozen. Of course, where the peptides are stored in alyophilized or powdered state, they may be stored virtuallyindefinitely, e.g., in metered aliquots that may be rehydrated with apredetermined amount of water (preferably distilled) or buffer prior touse.

Pharmaceutical Compositions

The vaccine and booster peptide compositions can be formulated asfreeze-dried or liquid preparations according to any means suitable inthe art. Peptide compositions comprising the E39 or E39′ peptideantigens can be prepared using techniques well known to those skilled inthe art including, but not limited to, mixing, sonication andmicrofluidation.

The peptide compositions can be formulated in either neutral or saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the active polypeptides) and whichare formed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or organic acids such as acetic, oxalic, tartaric,mandelic, and the like. Salts formed from free carboxyl groups can alsobe derived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,procaine, and the like.

In general, the peptide compositions typically comprise apharmaceutically acceptable carrier. As used herein, the term“pharmaceutically acceptable” means approved by a government regulatoryagency or listed in the U.S. Pharmacopeia or another generallyrecognized pharmacopeia for use in animals, particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the compound is administered.

In certain embodiments, the peptide compositions are provided in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the subjects to be treated; each unit contains apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier.

In one embodiment, the peptide compositions are formulated forinoculation or injection, for example, either subcutaneously(intradermally) or intramuscularly into the subject.

Non-limiting examples of liquid form preparations include solutions,suspensions, syrups, slurries, and emulsions. Suitable liquid carriersinclude any suitable organic or inorganic solvent, for example, water,alcohol, saline solution, buffered saline solution, physiological salinesolution, dextrose solution, propylene glycol solutions, water-in-oilemulsions, biodegradable oil vehicles, oil-in-water emulsions, liposomeemulsions, and the like, and combinations thereof, preferably in sterileform.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofdimethyl sulfoxide (DMSO) as solvent is envisioned to result inextremely rapid penetration, delivering high concentrations of theactive agents to a small area.

In one embodiment, the peptide compositions are provided inbacteriostatic water. In another embodiment, the peptide compositionsare stored at or near 0° C. and thawed prior to use.

In alternative embodiments, the vaccine compositions can also beformulated in sustained release vehicles or depot preparations. Suchlong acting formulations can be administered by inoculation orimplantation (for example subcutaneously or intramuscularly) or byinjection. Thus, for example, the vaccine compositions can be formulatedwith suitable polymeric or hydrophobic materials (for example, as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salt. Liposomesand emulsions are well-known examples of delivery vehicles suitable foruse as carriers.

In addition, if desired, the peptide compositions may contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, bacteriostatic agents and/or antioxidants. Suitableagents which prevent the action of microorganisms can be brought aboutby preservatives such as various antibacterial and antifungal agents,including but not limited to parabens (e.g, methylparabens,propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal orcombinations thereof. Suitable preservatives for use in such a solutioninclude benzalkonium chloride, benzethonium chloride, chlorobutanol,thimerosal and the like. Suitable buffers include boric acid, sodium andpotassium bicarbonate, sodium and potassium borates, sodium andpotassium carbonate, sodium acetate, sodium biphosphate and the like, inamounts sufficient to maintain the pH at between about pH 6 and pH 8,and preferably, between about pH 7 and pH 7.5. Suitable tonicity agentsare dextran 40, dextran 70, dextrose, glycerin, potassium chloride,propylene glycol, sodium chloride, and the like, such that the sodiumchloride equivalent of the ophthalmic solution is in the range 0.9±0.2%.Suitable antioxidants and stabilizers include sodium bisulfite, sodiummetabisulfite, sodium thiosulfate, thiourea and the like. Suitablewetting and clarifying agents include polysorbate 80, polysorbate 20,poloxamer 282 and tyloxapol. Suitable viscosity-increasing agentsinclude dextran 40, dextran 70, gelatin, glycerin,hydroxyethylcellulose, hydroxmethyl-propylcellulose, lanolin,methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol,polyvinylpyrrolidone, carboxymethylcellulose and the like.

Biological Response Modifiers

In some embodiments, it may be desirable to coadminister biologicalresponse modifiers (BRMs), which have been shown to upregulate T cellimmunity or down-regulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA);low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, NJ), cytokinessuch as γ-interferon, IL-2, or IL-12 or genes encoding proteins involvedin immune helper functions, such as B-7.

Chemokines, nucleic acids that encode for chemokines, and/or cells thatexpress such also may be used as vaccine components. Chemokinesgenerally act as chemoattractants to recruit immune effector cells tothe site of chemokine expression. It may be advantageous to express aparticular chemokine coding sequence in combination with, for example, acytokine coding sequence, to enhance the recruitment of other immunesystem components to the site of treatment. Such chemokines include, forexample, RANTES, MCAF, MIP1-alpha, MIP1-Beta, IP-10 and combinationsthereof. The skilled artisan will recognize that certain cytokines arealso known to have chemoattractant effects and could also be classifiedunder the term chemokines.

In one embodiment, the methods of the invention further includeadministration of an immunostimulatory cocktail called COVAX, consistingof a Toll-like receptor 7 (TLR7) agonist (e.g., imiquimod cream), anagonistic anti-CD40 antibody, and interleukin-2 (IL-2).

Patient Selection

Provided herein are effective methods for treating patients having anendometrial or ovarian cancer. In certain embodiments, the endometrialcancer is a serous variant endometrial cancer. In another embodiment,the cancer is ovarian cancer. In another particular embodiment, theendometrial cancer is serous uterine carcinoma.

In some embodiments, the endometrial or ovarian cancer expresses a lowlevel of FRα. Art recognized techniques for determining the FRα proteinor nucleic acid expression levels. For example, the tumor can bebiopsied and protein or nucleic acid expression level of FRα determinedby immunohistochemistry or fluorescence in situ hybridization using aspreviously described (e.g., Shia et al. Human Pathol. 39:498-505, 2008;Leung et al. Clin. Biochem 46; 1462-1468, 2013; O'Shannessy et al.OncoTarget. 2:1227-1243, 2011).

In certain embodiments, the endometrial or ovarian cancer has an IHCrating of 0 or 1+ for FRα protein expression. The IHC rating of FRα isdetermined according to known techniques (e.g., Markert et al.Anticancer Res. 28:3567-3572, 2008).

In certain embodiments, the patient has been diagnosed with a primarycancer, i.e., the cancer is not a recurrence of previously diagnosedtumor after prior treatment.

In related embodiments, the patient has no evidence of disease followingat least one chemotherapy-containing regimen that is considered standardof care for that cancer type.

In another embodiment, the patient meets one or more of the followingclinical criteria:

-   -   (1) no evidence of disease (NED) after completion of primary        first-line therapies (i.e., surgery, chemotherapy, immunotherapy        and radiation therapy as appropriate per standard of care for        patient's specific cancer);    -   (2) post-menopausal or rendered surgically infertile;    -   (3) good performance status (Karnofsky>60%, ECOG≤2), CBC, CMP        and A-125.

Treatment Regimens

The primary immunization schedule for a subject is sufficient to induceand/or sustain a therapeutically effective immune response to FRα. Forexample, a therapeutically effective amount will provide a clinicallysignificant increase in the number of E39-specific cytotoxicT-lymphocytes (CD8⁺) in the subject, as well as a clinically significantincrease in the cytotoxic T-lymphocyte response to the FRα antigen, asmeasured by any means suitable in the art. A therapeutically effectiveamount can also destroy residual microscopic disease and significantlyreduce or eliminate the risk of cancer recurrence in the subject.

Suitable treatment protocols or regimens for inducing therapeuticimmunity against an endometrial tumor characterized as having low FRαexpression (IHC of 0 to 1+) include administering to the subject apeptide vaccine comprising about 1.0 mg of peptide consisting of theamino acid sequence of SEQ ID NO: 1 and an adjuvant (GM-CSF) every threeto four weeks for a period of at least about six months.

In certain embodiments, the primary immunization schedule comprises aperiod of about six months in which a vaccine comprising 1 mg of the E39peptide and 0.250 mg of GM-CSF is administered every three to four weeksas a split inoculation by concurrent injections about 5 cm apart on thepatient's body, for a total of six doses during a six month period.

In certain embodiments, the primary immunization schedule is startedafter completion of prior treatment according to the accepted standardof care for the cancer type, including, but not limited to surgery,radiation, targeted therapy, chemotherapy, growth factor inhibitors,anti-angiogenesis factors or a combination of one or more thereof. Insome embodiments, the subject will have received surgical resection ofthe endometrial tumor, first-line platinum-based therapy, first-linetaxane-based therapy or a combination thereof prior to treatment withthe E39 peptide vaccines. In some embodiments, a subject receivessurgery, platinum, taxane, and avastin.

In particular embodiments, the primary immunization schedule is startedabout 4 months, 3 months, 2 months or less after the prior cancertreatment. In one embodiment, the primary immunization schedule isstarted no more than three months after completion of prior cancertreatment.

In other aspects, the methods further provide administration of abooster composition once the primary immunization schedule has beencompleted to bolster and/or sustain the therapeutic immunity to FRα.Booster compositions contain a peptide comprising the amino acidsequence of E39 (SEQ ID NO: 1) or E39′ (SEQ ID NO: 2) and an adjuvant.The booster composition can be administered at regular intervals ofabout 3, about 6, about 9 or about 12 months after completion of theprimary immunization schedule. In a particular embodiment, the boostercomposition is administered about every 6 months. In an alternativeembodiment, boosters can be administered on an as-needed basis, forexample, based on when there is a decrease in the FRα immune response(as measured, e.g, by CTL or antibody titers), or an increase incirculating FRα-expressing tumor cells in the subject.

Administration of the peptide vaccine and booster compositions can be byinfusion or injection (e.g., intravenously, intramuscularly,intracutaneously, subcutaneously, intrathecally, intraduodenally,intraperitoneally, and the like). The peptide vaccine compositions canalso be administered intranasaly, vaginally, rectally, orally ortransdermally. In a particular embodiment, the peptide vaccinecompositions are administered by intradermal injection.

In some embodiments, each dose of the peptide vaccine and boostercompositions can be split into multiple injections, which areadministered preferably substantially concurrently. When administered asa split inoculation, the dose of the peptide vaccine or boostercomposition is preferably, but not necessarily, proportioned equally ineach separate injection. In addition, the dose of the adjuvant ispreferably, but not necessarily, proportioned equally in each separateinjection. The separate injections for the split inoculation arepreferably administered substantially proximal to each other on thepatient's body. In one embodiment, the injections are administered atleast about 1 cm apart from each other on the body. In anotherembodiment, the injections are administered at least about 2.5 cm apartfrom each other on the body. In a particular embodiment, the injectionsare administered at least about 5 cm apart from each other on the body.In another embodiment, the injections are administered at least about 10cm apart from each other on the body. In other embodiments, theinjections are administered more than 10 cm apart from each other on thebody, for example, at least about 12.5, 15, 17.5, 20 cm or more apartfrom each other on the body. Primary immunization injections and boosterinjections can be administered as a split inoculation as described andexemplified herein.

The booster compositions used in the methods provided herein compriseabout 0.1 to about 2.0 mg of either the E39 or E39′ peptide. In oneembodiment, the booster composition comprises about 500 mcg, about 600mcg, 700 mcg, 800 mcg, 900 mcg or 1000 mcg of the E39 peptide. Inanother embodiment, the booster composition comprises about 500 mcg,about 600 mcg, 700 mcg, 800 mcg, 900 mcg or 1000 mcg of the E39′peptide. In one preferred embodiment, the booster composition comprisesabout 500 mcg or about 1000 mcg of the E39 peptide. In a particularembodiment, the booster composition contains about 500 mcg or about 1000mcg of the E39′ peptide.

In some embodiments, the adjuvant in the vaccine and booster compositioncan comprise from about 10% to about 50% (v/v), preferably about 20% toabout 40% (v/v), and more preferably about 20% to about 30% (v/v) of thevaccine and booster compositions. In certain embodiments, the adjuvantis granulocyte macrophage-colony stimulating factor (GM-CSF). In onepreferred embodiment, the vaccine and/or booster compositions comprisefrom about 0.01 mg to about 0.5 mg of GM-CSF. In another preferredembodiment, the vaccine and/or booster compositions comprise about 0.125mg of GM-CSF. In another preferred embodiment, the vaccine and/orbooster compositions comprise about 0.250 mg of GM-CSF.

After a period of about 48 hours after each dose of the vaccinecomposition is administered, the injection site can be assessed forlocal reaction of erythema and induration. If the reactions at bothsites are confluent and the area of total induration measures >100 mm(or the patient experiences any >grade 2 systemic toxicity), then thedose of GM-CSF can be reduced, for example, by half, though it isintended that the peptide dose remain the same. If the patient presentsa robust reaction on subsequent doses, then further reduction of GM-CSFcan occur, for example, reducing by half. If the patient does notpresent with a robust reaction, then the patient can continue with thehigher GM-CSF dose.

In other embodiments, the administration schedule and dosing of thebooster is similarly determined, with boosters beginning withadministration of vaccine compositions comprising 1 mg of E39 or E39′and 0.25 mg GM-CSF, administered about every six months following theconclusion of the primary immunization vaccine schedule.

Therapeutic Response

Subjects treated according to the methods disclosed herein mayexperience improvement in at least one sign or symptom associated withendometrial cancer.

In one embodiment, the subject experiences tumor shrinking and/ordecrease in growth rate, i.e., suppression of tumor growth. In anotherembodiment, recurrence of the tumor is prevented or delayed. In relatedembodiments, one or more of the following can occur: the number ofcancer cells can be reduced; tumor size can be reduced; cancer cellinfiltration into peripheral organs can be inhibited, retarded, slowed,or stopped; tumor metastasis can be slowed or inhibited; tumor growthcan be inhibited; and progression free survival (PFS) is increased.

In some embodiments, therapeutic response is measured by a reduction inthe quantity and/or size of measurable tumor lesions. Measurable lesionsare defined as those that can be accurately measured in at least onedimension (longest diameter is to be recorded) as ≥10 mm by CT scan (CTscan slice thickness no greater than 5 mm), 10 mm caliper measurement byclinical exam or >20 mm by chest X-ray. The size of non-target lesions,e.g., pathological lymph nodes can also be measured for improvement. Inone embodiment, lesions can be measured on chest x-rays or CT or MRIfilms.

In other embodiments, cytology or histology can be used to evaluateresponsiveness to a therapy. The cytological confirmation of theneoplastic origin of any effusion that appears or worsens duringtreatment when the measurable tumor has met criteria for response orstable disease can be considered to differentiate between response orstable disease (an effusion may be a side effect of the treatment) andprogressive disease.

In yet another embodiment, therapeutic response is measured by adecrease in recurrence rate of the endometrial tumor in the subject whencompared to the average recurrence rate in untreated control subjects.Recurrence of lack thereof can be expressed as disease-free survival(DFS) or recurrence-free survival (RFS) using clinically acceptablecriteria.

In other embodiments, the subject experiences one or more of thefollowing: a clinically significant increase in FRα-CTL activity; aclinically significant decrease in serum FRα levels; a clinicallysignificant decrease serum CA125 levels; and a clinically significantdecrease in circulating tumor cells (CTC) expressing FRα.

Exemplary therapeutic responses to therapy may include:

-   -   Disease Free Survival (DFS): At least a 10% delay in the time to        recurrence of detectable disease;    -   Partial Response (PR): At least a 30% decrease in the sum of        dimensions of target lesions, taking as reference the baseline        sum diameters;    -   Stable Disease (SD): Neither sufficient shrinkage to qualify for        partial response, nor sufficient increase to qualify for        progressive disease, taking as reference the smallest sum        diameters while on study; or    -   Complete Response (CR): Disappearance of all non-target lesions        and normalization of tumor marker level. All lymph nodes must be        non-pathological in size (<10 mm short axis).    -   Non-CR/Non-PD refers to a response presenting a persistence of        one or more non-target lesion(s) and/or maintenance of tumor        marker level above the normal limits.

Progressive Disease (PD) refers to a response presenting at least a 20%increase in the sum of dimensions of target lesions, taking as referencethe smallest sum on study (this includes the baseline sum if that is thesmallest on study). In addition to the relative increase of 20%, the summust also demonstrate an absolute increase of 5 mm. The appearance ofone or more new lesions is also considered progression.

In some embodiments, an effective amount of the compositions providedherein produce at least one therapeutic effect selected from the groupconsisting of reduction in size of a FRα-expressing tumor, reduction inmetastasis, complete remission, partial remission, stable disease,increase in overall response rate, a pathologic complete response or adelay in tumor recurrence. In other embodiments, the improvement ofclinical benefit rate is about 10%-20%, 30%, 40%, 50%, 60%, 70%, 80% ormore.

Combination Treatments

The present methods can be combined with other means of cancer treatmentincluding, but not limited to surgery, radiation, targeted therapy,chemotherapy, growth factor inhibitors, anti-angiogenesis factors or acombination of one or more thereof.

In particular embodiments, the patient receives treatment with one ormore cancer therapies at least 12 hours, one day, a week, a month, sixweeks, two months or three months prior to administration of the E39peptide vaccine composition. In another embodiment, during the course ofadministration of the E39 and/or E39′ peptide vaccines, a patient istreated concurrently with one or more additional cancer therapies.

In other embodiments, the patient may receive one or more additionalcancer therapies subsequent to administration of the E39 peptide vaccinecomposition. For example, vaccination against FRα-specific antigens maysensitize the endometrial tumor against subsequent chemotherapeutictreatment. Without being bound by any one theory, it is possible thatchemotherapy-induced systemic effects (e.g., enhanced cross-presentationof tumor antigens, depletion of suppressor cells, activation of DCs) mayfurther promote the antitumor activity of FRα-specific CTLs.

In other embodiments, the additional cancer therapy is ananti-angiogenisis targeted therapy such as Bevacizumab (Avastin®),Aflibercept (VEGF Trap), Cediranib (Recentin™), Nintedanib (BIBF 1120),Pazopanib, Sorafenib (Nexavar®), Sunitinib (Sutent®), and Cabozantinib(XL-184); Ribose Polymerase (PARP) inhibitors, such as Olaparib(AZD2281), Iniparib (BSI-201), MK04827, ABT-888 and BMN-673;Phosphatidylinositol-3-kinase (PI3K)/Protein Kinase B (AKT)/mTOR pathwayinhibitors, such as XL147, PX-866, Everolimus (Afinitor®), Temsirolimus(Torisel®) and Perifosine, MK-2206; SRC inhibitors such as Dasatinib(Sprycel®), and Saracatinib (AZD0530); EGFR Inhibitors such as cetuximab(Erbitux®), trastuzumab (Herceptin®), pertuzumab, erlotinib (Tarceva®),MM-121, and vandetanib; EGFRα inhibitors such as Farletuzumab(MORAb-003), EC145 (conjugate of folic acid and desacetylvinblastine);IGF inhibitors such as AMG 479 and dalotuzumab; p53 inhibitors such asMK-1775; and cytotoxic agents such as cyclophosphamide, carboplatin,pegylated liposomal doxorubisin (PLD), gemcitabine, paclitaxel,topotecan.

In some embodiments, the methods further include administration of atherapeutically effective amount of a platinum-containing compoundand/or a taxane to the subject. Exemplary platinum-containing compoundsinclude, but are not limited to, cisplatin and carboplatin. Theplatinum-containing compound may be administered to the subject aboutonce every week, about once every two weeks, about once every threeweeks, or about once every four weeks.

Examples of taxanes include, but are not limited to, paclitaxel,docetaxel, and semi-synthetic, synthetic, and/or modified versions andformulations thereof, including but not limited to nab-paclitaxel(Abraxane®), cabazitaxel (Jevtana®), DJ-927 (Tesetaxel®), paclitaxelpoliglumex (Opaxoi®), XRP9881 (Larotaxel®), EndoTAG+pacitaxel(EndoTAG®-1), polymeric-micellar paclitaxel (Genexol-PM®),DHA-paclitaxel (Taxoprexin®), BMS-184476. The taxane may be administeredto the subject once every week, about once every two weeks, about onceevery three weeks, or about once every four weeks. In embodiments inwhich both a platinum-containing compound and a taxane are administeredto the subject prior to or as part of the treatment regimen, theplatinum-containing compound may be administered before, after orsimultaneously with the taxane.

Kits and Articles of Manufacture

Further provided are kits containing the peptide vaccine compositionsdescribed herein and instructions for use. Kits typically include apackaged combination of reagents in predetermined amounts withinstructions and a label indicating the intended use of the contents ofthe kit. The term label or instruction includes any writing, or recordedmaterial supplied on or with the kit, or which otherwise accompanies thekit at any time during its manufacture, transport, sale or use. It canbe in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of the manufacture, use orsale for administration to a human or for veterinary use. The label orinstruction can also encompass advertising leaflets and brochures,packaging materials, and audio or video instructions.

For example, in some embodiments, the kit contains the peptide vaccinein a suitable container and instructions for administration. In someembodiments, the peptide vaccine is provided in a suitable container asa dosage unit for administration. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers may beformed from a variety of materials such as glass or plastic.

In other embodiments, the peptide vaccine is provided in lyophilizedform, and the kit may optionally contain a sterile and physiologicallyacceptable reconstitution medium such as water, saline, buffered saline,and the like. It may further include other materials desirable from acommercial and user standpoint, including other buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse, for example, comprising administration schedules, to allow apractitioner (e.g., a physician, nurse, or patient) to administer thecomposition contained therein.

INCORPORATION BY REFERENCE

All documents and references, including patent documents and websites,described herein are individually incorporated by reference to into thisdocument to the same extent as if there were written in this document infull or in part.

The following examples are merely illustrative and should not beconstrued as limiting the scope of this disclosure in any way as manyvariations and equivalents will become apparent to those skilled in theart upon reading the present disclosure.

Example 1 Response to E39 in Patients with Endometrial or Ovarian Cancer

The purpose of this study was to test whether a peptide-based vaccineconsisting of the E39 peptide mixed with the FDA-approved immunoadjuvantgranulocyte macrophage colony-stimulating factor (GM-CSF) is safe andeffective at inducing an in vivo peptide-specific immune response.

Methods Patient Characteristics and Clinical Protocols

Eligible patients were identified with endometrial, ovarian, fallopiantube or peritoneal cancer and were disease-free after standard of caretherapies. Patients were surgically or naturally post-menopausal.Exclusion criteria included patients currently receivingimmunosuppressive therapy to include chemotherapy, steroids ormethotrexate, poor health (ECOG<2), evidence of end-organ dysfunction,pregnancy, breast feeding, history of autoimmune disease, andinvolvement in other experimental protocols (except with permission ofthe principal investigator of the other study). Exclusion criteriaincluded patients currently receiving immunosuppressive therapy toinclude chemotherapy, steroids or methotrexate, poor health (ECOG<2),evidence of end-organ dysfunction, pregnancy, breast feeding, history ofautoimmune disease, and involvement in other experimental protocols.

Once enrolled, HLA status was evaluated to permit group assignment. E39is a HLA-A2-restricted peptide (HLA-A2 is present in approximately40-50% of the general population). HLA-A2 positive patients were thusinoculated with E39+GM-CSF, while HLA-A2 negative patients and thoseHLA-A2 positive individuals who declined vaccination were followedprospectively as matched controls for disease recurrence andprogression. The clinical endpoints were long-term FBP immunity, time torecurrence from date of enrollment, and 2-year disease-free survival(DFS) rate. This planned interim analysis was performed 12 months aftercompletion of trial enrollment.

Fifty-one (51) patients were enrolled in the study with twenty-nineHLA-A2+ patients in the vaccination group (VG), and twenty-two HLA-A2−patients as a control group (CG). The There were no significantdifferences in age, grade, stage, or histology between groups (allp>0.1). Demographic, safety, immunologic, and clinical recurrence (CR)data were collected. Disease free survival (DFS) was compared byKaplan-Meir and logrank tests. Continuous variables were compared withanalysis of variance and proportions with Fisher's exact test.

Vaccine and Vaccination Series

The E39 peptide (FBP 191-199, EIWTHSYKV) was produced commercially by anFDA-compliant production facility for patient use. The peptide waspurified to >95% before use. Sterility, endotoxin (limulus amebocytelysate test), and general safety testing was performed. In addition, themanufacturer performed purity/stability testing periodically. Singledose vials were tested for bacterial and fungal contaminants prior touse. The single dose vials were stored in the pharmacy at eachinstitution. The bulk peptide was reconstituted to the followingpreparations: 100 mcg/0.5 mL, 500 mcg/0.5 mL, and 1000 mcg/0.5 mL. Eachof these was mixed with 250 mcg/1.0 mL GM-CSF. This dose (250 mcg) ofGM-CSF has been previously determined to be a safe and effective dose,based on our prior work with NeuVax⁶.

The combination of peptide and adjuvant had a volume of 1.5 mL, whichwas administered intradermally in 0.75 mL inoculums at two differentsites within 5 cm of each other. The primary vaccination series (PVS)consisted of six total vaccinations, one given every 21-28 days,administered in the same lymph node draining area.

Dosing

The phase I portion of this trial consisted of dose escalation todetermine a safe and effective dose of the E39 peptide. The doseescalation scheme consisted of dosing cohorts of three patientsreceiving one the following doses: 100, 500, and 1,000 mcg of peptide,in addition to 250 mcg of GM-CSF. Prior to the fourth inoculation, eachpatient was assessed for liver, renal, and hematopoietic dysfunction. Iforgan function was stable and no dose limiting toxicity was observed,then the patient continued the series. After the third patient in agiven dose group completed the third inoculation and organ functionremained stable, the next dose group was initiated.

The optimal dose was initially defined immunologically, but wasredefined when the 1000 mcg dosing cohort showed clinical benefit ininitial survival data. This cohort was then expanded and became known asthe optimal dose group (OPT). The phase IIa portion of this trial thenanalyzed the expanded OPT versus all other patients.

Toxicity

Standard local and systemic toxicities were collected and graded per theNational Cancer Institute Common Terminology Criteria for AdverseEvents, v4.03 toxicity scale. For the vaccine series (one vaccine/monthfor six months), patients were monitored closely for one hour aftervaccine inoculation with questioning, serial exams and vital signs every15 minutes to observe for a hypersensitivity reaction. Systemic toxicityand inoculation site local reactions were also determined after 48-72hours.

In Vivo Immune Monitoring

Patients were assessed for evidence of in vivo immunologic response byevaluation of delayed-type hypersensitivity (DTH) reaction, which wasmeasured pre-vaccination and again post-PVS. A DTH response was assessedwith 100 mcg of E39 (without GM-CSF) as well as a parallel control(equivalent volume of sterile saline) injected intradermally at a siteon the back or anterior thigh on the opposite side from the vaccinationsite. The response was measured using the sensitive ballpoint-pen methodin 2 dimensions at 48-72 hours post injection¹⁵. The orthogonal mean wasdetermined for each DTH; its correlation to immunologic response hasbeen previously validated and used in our previous work¹⁶. These valueswere compared between pre-PVS and post-PVS.

Clinical Recurrences of Disease

Both the vaccinated patients and the observational control patients weremonitored for evidence of clinical recurrence through the standard ofcare follow-up with their primary medical and/or surgical oncologist.This consisted of evaluations every three-months for the initial twoyears, then every six-months for an additional three years with clinicalexam, laboratory tests and radiographic surveillance as indicated.Patients' clinical records were assessed for evidence of clinicalrecurrence. Disease free survival was measured from the date ofenrollment. All patients were followed for clinical recurrence for up totwo years at standard of care visits.

Statistical Analysis

A pre-specified, intention-to-treat analysis was performed at 12 monthsafter the last patient was enrolled. Clinicopathologic data werecompared between groups. Median and range were used to summarizecontinuous data and the groups were compared using a Mann-Whitney Utest. Chi squared or Fischer exact test were used to compare categoricalvariables between groups. DTH data was presented as orthogonalmeans±standard errors and compared using a Student's t test. Time torecurrence was measured from the date of enrollment. DFS was analyzedusing the Kaplan-Meier method, and groups were compared using a simplelog-rank test. Statistical analyses were performed using SPSS version 22(IBM Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0.Armonk, N.Y.: IBM Corp.). Statistical significance was consideredachieved if p<0.05. Pre-specified subset analyses were also performed bydose cohort.

Results Patients

Fifty-one (51) patients were enrolled in the study, 29 in the vaccinatedgroup (VG) and 22 patients in the control group (CG). Within the VG, 15patients received the optimal dose (1000 mcg) of the peptide, referredto as the OPT group, while 14 patients received less, referred to as thenon-optimal dose (nOPT) group (FIG. 1). There were no significantclinicopathologic differences between groups (Table 1A and 1B).

TABLE 1A Demographics Vaccinated Controls Characteristic (n = 29) (n =22) p-Value Median age (yrs) 60 61 0.790 (Interquartile Range 1-3) 52-6753-63 Histology- n (%) 0.373 Endometrial 6 (20.7) 3 (13.6) Fallopian 1(3.4) 0 (0.0) Ovarian 20 (69.0) 18 (86.4) Peritoneal 2 (6.9) 0 (0.0)Grade- n (%) 0.851 1 (Well Differentiated) 2 (6.9) 2 (9.1) 2 (ModeratelyDifferentiated) 4 (13.8) 2 (9.1) 3 (Poorly Differentiated) 23 (79.3) 18(81.8) Stage- n (%) 0.235 Tis 1 (3.4) 0 (0.0) 1 6 (20.7) 3 (13.6) 2 2(6.9) 4 (18.2) 3 14 (48.3) 12 (54.5) 4 5 (17.2) 3 (13.6) Tx 1 (3.4) 0(0.0) Node- n (%) 0.764 Positive 9 (31.0) 5 (22.7) Negative 20 (69.0) 17(77.3) FIGO Stage- n (%) 0.591 I 4 (13.8) 3 (13.6) II 2 (6.9) 3 (13.6)III 18 (62.1) 11 (50.0) IV 5 (17.2) 5 (22.7)

TABLE 1B Demographics - Dosing Cohorts <1000 mcg 1000 mcg (nOPT) (OPT)Controls Characteristic (n = 14) (n = 15) (n = 22) p-Value Median age(yrs) 61 57 61 0.827 (Interquartile range 1-3) 56-66 49-67 53-63Histology- n (%) 0.531 Endometrial 3 (21.4) 3 (20.0) 3 (13.6) Fallopian1 (7.1) 0 (0.0) 0 (0.0) Ovarian 9 (64.3) 11 (73.3) 18 (86.4) Peritoneal1 (7.1) 1 (6.7) 0 (0.0) Grade - n (%) 0.508 1 (Well Differentiated) 0(0.0) 2 (13.3) 2 (9.1) 2 (Moderately Differentiated) 3 (21.4) 1 (6.7) 2(9.1) 3 (Poorly Differentiated) 11 (78.6) 12 (80.0) 18 (81.8) T Stage- n(%) 0.263 Tis 0 (0.0) 1 (6.7) 0 (0.0) 1 3 (21.4) 3 (20.0) 3 (13.6) 2 0(0.0) 2 (13.3) 4 (18.2) 3 9 (64.3) 5 (33.3) 12 (54.5) 4 2 (14.3) 3(20.0) 3 (13.6) Tx 0 (0.0) 1 (6.7) 0 (0.0) Node- n (%) 0.450 Positive 6(42.9) 3 (20.0) 5 (22.7) Negative 8 (57.1) 12 (80.0) 17 (77.3) FIGOStage- n (%) 0.297 I 1 (7.1) 3 (20.0) 3 (13.6) II 0 (0.0) 2 (13.3) 3(13.6) III 11 (78.6) 7 (46.7) 11 (50.0) IV 2 (14.3) 3 (20.0) 5 (22.7)

Toxicity

Local and systemic toxicities were mild at the completion of the PVS(FIG. 2), with no grade 4 or 5 toxicities, and only 1 patientexperiencing grade 3 toxicity. The maximum local toxicities were milderfor the OPT patients than nOPT patients (p=0.04) with OPT and nOPTgroups experiencing grade 1 (100% vs 78.6%) or grade 2 (0% vs 21.4%).The most common local toxicities were induration at the injection site,erythema, and pruritus. The maximum systemic toxicities for the OPTgroup as compared to the nOPT group were grade 0 (0% vs 7.7%), grade 1(61.5% vs 46.2%), grade 2 (38.5% vs 46.2%) and grade 3 (0% vs 7.7%),(p>0.05 for each). The most common systemic toxicities were myalgias,headache, and fatigue (FIG. 1).

Immunologic Response

The DTH increased from pre-vaccination to post-vaccination in the VG,approaching statistical significance (5.7±1.5 mm vs 10.3±2.8 mm,p=0.06). As shown in FIG. 3, when analyzed by dose, OPT patients had astatistically significant increase in DTH from pre-vaccination topost-vaccination (3.8±2.0 mm vs 9.5±3.5 mm, p=0.03), while nOPT patientsexperienced a smaller increase from pre- to post-vaccination, which wasnot statistically significant (7.8±2.1 mm vs 11.3±4.8 mm, p=0.28).

Disease-Free Survival

An interim analysis was performed 12 months after completion of trialenrollment. The median follow up was 12.0 months (interquartile range7.6-19.2 months). The overall recurrence rate for the VG versus the CGwas 41.4% versus 54.6%, respectively (p=0.35). The 2-year estimated DFSfor VG was 43% (95% confidence interval (CI): 18-66%) versus 33.6% (95%CI: 13-56%) in CG (p=0.36). The vaccinated patients experienced a 31%reduction in relative recurrence risk regardless of dose (FIG. 4).

Subgroup analyses were performed based on dosing cohort. The recurrencerate was significantly lower in the OPT group compared to the CGpatients (13.3% vs 54.6%, p=0.02). There was no statisticallysignificant difference in recurrence between the nOPT group versus theCG patients. When comparing the dosing cohorts, patients receiving theoptimal dose of E39 experienced an 83% reduction in relative risk ofrecurrence compared to nOPT patients (HR 0.17, 95% CI: 0.04-0.77,p=0.003). The 2-year estimated DFS indicated a significant survivaladvantage for the OPT group at 85.7% (95% CI: 54-96%) compared to thecontrols at 33.6% (95% CI: 13-56%, p=0.02). Surprisingly, there was nodifference of statistical significance in the estimated 2-year DFSbetween the nOPT and CG. A survival benefit was appreciated withanalysis of the estimated 2-year DFS comparing OPT versus the nOPTpatients, 85.7% versus 20.8% (p=0.009). FIG. 5 displays the survivalanalysis of a 3-way comparison between OPT, nOPT and CG.

Discussion

This trial using E39+GM-CSF to prevent recurrence in disease-freeendometrial and ovarian cancer patients at high risk of recurrencedemonstrated that the vaccine is well tolerated, immunogenic. No grade 4or 5 toxicities were observed. The only grade 3 response was observed inthe nOPT cohort, indicating the increase in peptide dose did not causeany additional toxicity. Given the severity of side effects associatedwith standard of care chemotherapy for endometrial and ovarian cancerpatients, a low toxicity profile is key for any additional therapy givenin the adjuvant setting. The E39 vaccine, even given at higher peptidedose, fulfills this requirement.

The data further demonstrate that the E39 vaccine was able toeffectively induce a robust immune response. Patients did have a limitedDTH response to E39 prior to vaccination, which indicates previousexposure to FBP on the surface of their tumors. The vaccine, however,increased this DTH response in all vaccinated patients and increased theresponse to an even greater extent in patients receiving the optimal(1.0 mg) dose of the vaccine, reaching statistical significance. The OPTgroup had a greater increase in DTH from pre- to post-vaccination andthe best clinical outcomes.

In the subgroup analysis, optimally dosed patients had an even morepronounced benefit, reaching statistical significance when compared toboth the CG and nOPT group. Indeed, it was completely unexpected thatpatients receiving less than the 1.0 mg OPT dose did not demonstrate astatistically significant benefit when compared to the CG. The OPT groupwas approximately five times less likely to experience a recurrence whencompared to the CG. Clearly, the higher dose of peptide induced a moremeaningful response in this group of patients. In addition, the efficacyresults indicate that the 1000 mcg dose cohort had increased immuneresponses and longer disease-free survival rates.

Example 2 Effect of Booster Inoculation on Immune Response

Booster inoculations of peptide vaccines may improve disease-freesurvival (DFS) (e.g., U.S. Pat. No. 8,222,214), but repeated boostingcan theoretically lead to overstimulation and loss of vaccine-induced Tcells. Therefore, to assess the use of the attenuated E39′ peptide as abooster, E39-vaccinated patients described in Example 1 were randomizedto receive either E39 or E39′ as a booster to determine the effect onlong-term E39-specific immunity. Patients in the VG received sixintradermal E39 injections as described in Example 1 as a primaryvaccination schedule (PVS). At six months (B1) and at twelve months (B2)after completion of the PVS, patients received either 500 mcg of E39′and 250 mcg GM-CSF or 500 mcg E39 and 250 mcg of GM-CSF as a booster.

Local reactions (LR) were recorded 48-72 hours after each booster.Immune response was determined by measurement of E39 specific CTL levelsusing a Dextramer reagents in accordance with the manufacturer'sinstructions (Immudex Limited, Copenhagen, Denmark) as previouslydescribed (e.g., Tario et al. Cytometry B Clin. Cytom. 2015; 88B:6-20;Baba J. Transl Med. 2010; 8:84), at RO (baseline pre-vaccination), RC1(1 month after completion of PVS), RC6 (6 months after completion of PVSand before booster), RC7 (1 month after booster) and RC12 (6 monthsafter booster and 12 months after completion of PVS). Demographic,safety, immunological, and DFS data were collected and evaluated.

Seventeen patients were included in this portion of the study. For B1,nine patients received E39′ and eight patients received E39; and B2included seven patients in each group (E39 or E39′). There were nosignificant clinicopathological differences between groups.

The data from a patient receiving E39′ as a booster is exemplified inTable 2.

TABLE 2 Timepoint R0 RC1 RC6 RC7 RC12 E39 CTL Level 0.018 0.018 0.078.275 .275

An increase in E39-specific CTLs was not seen immediately aftercompletion of the PVS. However, there was a substantial increase by 6months after completion of the PVS (>4 fold). Even more significantly,the level of CTLS increased even further after boosting with the E39′peptide (>15 fold over baseline) which persisted for at least 6 months.

The average LR for all patients in the E39′ vs. E39 groups were79.7±14.0 mm vs. 82.1±8.3 mm, respectively for B1 (p=0.45) and 74.1±11.5vs. 78±11.2 mm, respectively for B2 (p=0.41). Clinically, the recurrencerate was 22.2% in the E39′ boost group vs. 25% for E39. The estimated2-year DFS for B1 pts for E39′, E39 and the control group (CG) were66.7%, 58.3%, and 36.0%, respectively; and for B2 pts were 66.7%, 66.7%,and 36.0%, respectively. Comparing just the boosted groups, for B1 thehazard ratio (HR) for E39′ vs. E39=0.71 (95% CI: 0.1-5.13), and for B2the HR for E39′ vs. E39=0.82 (95% CI: 0.05-13.24).

The data indicate that the use of an attenuated peptide (E39′) boosterwas safe and as immunogenic as the wildtype peptide (E39) in thisrandomized trial of optimal boosting strategies. More importantly, thedata further show that there appears to be a potential clinicaladvantage to the use of attenuated E39′ peptide booster.

Example 3 Clinical Outcomes and FBP Expression Levels

Disease-free HLA-A2+ patients were vaccinated (VG) and HLA-A2− followedas controls (CG). VG received 6-monthly inoculations of E39+GM-CSF. FBPexpression was graded 0-4 based on percentage positive staining; 0-1+categorized low expression (FBP_(lo)), 2-4+ categorized high expression(FBP_(hi)). Demographics, FBP expression and disease-free survival (DFS)were analyzed.

Thirty-eight enrolled patients underwent FBP expression testing(CG-n=20; VG-n=18). There were no clinicopathologic differences betweengroups (p>0.1). Nineteen patients were FBP_(lo) and 19 were FBP_(hi).Median follow up was 16.3 months. In FBP_(lo) patients there wasimproved DFS in VG (n=8) vs. CG (n=11; 85.7% vs. 17.5%, p=0.01). Therewas no difference in FBP_(hi) patients (VG:13.9% vs. CG:44.4%, p=0.83).Among FBP_(lo) patients, there was a dose-dependent effect on DFS withpatients receiving 1000 mcg having improved DFS vs. <1000 mcg and CG(100% vs. 66.7% vs. 17.5%, p=0.03). (FIGS. 6-10)

Surprisingly, this phase I/IIa revealed a DFS benefit in FBP_(lo), overFBP_(hi) E+OC patients treated with E39. This may be due toimmunotolerance in FBP_(hi) patients.

TABLE 3 Demographics Control Treatment n= 22 n= 29 Age at Median/Range61 47 61 37 0.754 enrollment Race and Asian 1 4.30% 1 3.40% EthnicityBlack 0 0.00% 1 3.40% Hispanic 1 4.30% 1 3.40% White 21 91.30% 26 89.70%0.837 Primary Endometrial 4 17.40% 7 24.10% Cancer Site Ovarian 1982.60% 20 69.00% Peritoneal 0 0.00% 1 3.40% Tubal 0 0.00% 1 3.40% 0.536FIGO Stage IA 3 13.00% 1 3.40% IB 1 4.30% 1 3.40% IC 0 0.00% 1 3.40% IIA0 0.00% 2 6.90% IIB 1 4.30% 0 0.00% IIC 3 13.00% 1 3.40% IIIA 0 0.00% 13.40% IIIB 3 13.00% 2 6.90% IIIC 7 30.40% 15 51.70% IV 4 17.40% 5 17.20%IVB 1 4.30% 0 0.00% 0.384 Grade G1 2 8.70% 1 3.40% G2 2 8.70% 2 6.90% G319 82.60% 26 89.70% 0.691 Primary Cancer 16 69.60% 22 75.90% RecurrentCancer 7 30.40% 7 21.40% 0.661

What is claimed is:
 1. A method of inducing an immune response to atumor characterized as having an IHC of 0 or 1+ for FRα proteinexpression in a human subject, the method comprising administering to asubject a peptide vaccine comprising about 1.0 mg of a peptidecomprising the amino acid sequence of SEQ ID NO: 1 (E39) and anadjuvant, every 3 to 4 weeks for a period of at least about six months.2. The method of claim 1, wherein the peptide vaccine is administered byinjection.
 3. The method of claim 1 or 2, wherein the peptide vaccine isadministered by intradermal injection.
 4. The method of any one ofclaims 1-3, wherein the peptide vaccine is administered as split dosagesthat are administered substantially concurrently.
 5. The method of claim4, wherein the split dosages are administered at one site or atdifferent sites.
 6. The method of claim 4 or 5, wherein the splitdosages are administered at least 5 cm apart.
 7. The method of any oneof claims 1-6, wherein the peptide vaccine comprises a peptideconsisting of the amino acid sequence of SEQ ID NO:
 1. 8. The method ofany one of claims 1-7, wherein the adjuvant is granulocytemacrophage-colony stimulating factor (GM-CSF).
 9. The method of claim 8,wherein the peptide vaccine comprises between about 0.01 to about 0.5 mgGM-CSF.
 10. The method of claim 8, wherein the peptide vaccine comprisesabout 0.250 mg GM-CSF.
 11. The method of any one of claims 1-10, whereinthe tumor is a an endometrial cancer or ovarian cancer.
 12. The methodof any one of claims 1-11, further comprising administering to thesubject a booster composition an adjuvant about six months, about twelvemonths or about one year after the primary immunization schedule iscompleted, wherein the booster composition comprises an effective amountof a peptide comprising an amino acid selected from the group consistingof SEQ ID NO: 1 and SEQ ID NO: 2, and an adjuvant.
 13. The method ofclaim 12, wherein the peptide in the booster composition comprises theamino acid sequence of SEQ ID NO:
 1. 14. The method of any one of claims12-13, wherein the peptide in the booster composition comprises theamino acid sequence of SEQ ID NO:
 2. 15. The method of any one of claims12-14, wherein the booster composition comprises about 0.1 mg to about 2mg of the peptide.
 16. The method of any one of claims 12-15, whereinthe booster composition comprises about 0.5 to about 1.0 mg of thepeptide.
 17. The method of any one of claims 12-16, wherein the adjuvantin the booster composition is GM-CSF.
 18. The method of claim 17,wherein the booster composition comprises about 0.01 mg to about 0.5 mgGM-CSF.
 19. The method of any one of claims 17-18, wherein the boostercomposition comprises about 0.250 GM-CSF.
 20. The method of any one ofclaims 12-19, wherein the booster composition is administered about sixmonths after the completion of the primary immunization schedule. 21.The method of any one of claims 1-20, wherein the tumor is a serousvariant of an endometrial cancer.
 22. The method of any one of claims1-21, wherein the tumor is serous uterine cancer.
 23. The method of anyone of claims 1-22, wherein the subject has been previously treated withone or more cancer therapies.
 24. The method of any one of claims 1-23,wherein the subject has no evidence of disease (NED).
 25. The method ofany one of claims 1-24, wherein the tumor is not recurrent.
 26. Themethod of any one of claims 1-25, wherein the peptide vaccine isadministered within about three months after treatment with one or morecancer therapies selected from the group consisting of surgery,radiation, chemotherapy or a combination of one or more thereof.